NINJA connects the co-repressor TOPLESS to jasmonate signalling

Abstract

Jasmonoyl-isoleucine (JA-Ile) is a plant hormone that regulates a broad array of plant defence and developmental processes. JA-Ile-responsive gene expression is regulated by the transcriptional activator MYC2 that interacts physically with the jasmonate ZIM-domain (JAZ) repressor proteins. On perception of JA-Ile, JAZ proteins are degraded and JA-Ile-dependent gene expression is activated. The molecular mechanisms by which JAZ proteins repress gene expression remain unknown. Here we show that the Arabidopsis JAZ proteins recruit the Groucho/Tup1-type co-repressor TOPLESS (TPL) and TPL-related proteins (TPRs) through a previously uncharacterized adaptor protein, designated Novel Interactor of JAZ (NINJA). NINJA acts as a transcriptional repressor whose activity is mediated by a functional TPL-binding EAR repression motif. Accordingly, both NINJA and TPL proteins function as negative regulators of jasmonate responses. Our results point to TPL proteins as general co-repressors that affect multiple signalling pathways through the interaction with specific adaptor proteins. This new insight reveals how stress-related and growth-related signalling cascades use common molecular mechanisms to regulate gene expression in plants.

Figure 1. NINJA interacts with JAZ proteins

a, Gel separation of proteins co-purified with JAZ1-TAP from Arabidopsis cells, after mock-treatment (−JA) or elicitation (+JA). Arrows mark positions of proteins identified by MS. Lane M represents a molecular weight marker (in kDa). b–c, NINJA and JAZ interacting protein domains in Y2H assays. Drawings represent JAZ1, NINJA, and derivatives. Numbers indicate ending amino acids. Transformed yeasts were spotted in 10- and 100-fold dilutions on control (−2) or selective medium (−3). Controls are empty vectors. b, NINJA interaction with the TIFY motif of JAZ1. c, JAZ1 interaction with the C domain of NINJA.

Figure 2. NINJA negatively regulates JA signalling

a, Phenotypes of transgenic NINJA OE and NINJA KD Arabidopsis lines. Average primary root length of 9-day-old seedlings germinated on control medium, 10 or 50 μM JA. Data are means ± s.e.m for n ≥ 18 (**P ≤ 0.01, ***P ≤ 0.001, genotype × treatment, see Supplementary Table 2). b, Upregulation of MeJA responsive genes in NINJA KD plants. Heat maps, generated by the Expression Browser software (http://bar.toronto.ca), represent MeJA modulated expression over time (in hours) of genes significantly induced in NINJA KD plants. Scale bar indicates the log fold change.

Figure 3. NINJA interacts with TPL and functions as a transcriptional repressor

a, NINJA and TPL interacting protein domains in a Y2H assay. Drawings represent TPL, NINJA and derivatives. Y2H analysis was performed as in Fig. 1. b, JA phenotype of tpl-1. Root growth analysis was performed as in Fig. 2. c–d, Transactivation activity in tobacco protoplasts transfected with a PUAS-fLUC reporter construct, effector constructs fused or not with GAL4DBD, and a P35S-rLUC normalization construct. Data are means relative to the reference (black bar) ± s.e.m for n=8 (**P ≤ 0.01, *** P ≤ 0.001; ANOVA, Dunnett’s post hoc test). c, Functionality of the NINJA EAR repression motif. d, MYC2 activity is repressed by NINJA.

Figure 4. Model for a general function of TPL proteins in plant hormone signalling

a, In the absence of jasmonates, bHLH MYC factors interact with the Jas domain of JAZ proteins that interact through their TIFY motif with domain C of NINJA. The EAR motif of NINJA is essential for interaction with the TPL co-repressors. b, In the presence of (+)-7-iso-JA-L-Ile, JAZ proteins interact with the ubiquitin ligase SCF^COI1 leading to proteosomal JAZ degradation and subsequent release of the NINJA/TPL complex from the MYC factors and activation of jasmonate-responsive gene expression. c, Jasmonate and auxin pathways are built on similar signalling modules. d, NINJA interacts with other group-II TIFY proteins which might be recruited by yet unknown transcription factors. e, Interaction of the NINJA-related AFP proteins with ABI5 and TPL to regulate ABA responses.